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Abstract

Bacillus anthracis is the causative agent of the disease anthrax and poses a threat due to its potential to be used as a biological weapon. The spore form of this bacterium is an extremely resistant structure, making spore decontamination exceptionally challenging. During spore germination, nutrient germinants interact with Ger receptors, triggering a cascade of events. A crucial event in this process is degradation of the cortex peptidoglycan by germination-specific lytic enzymes (GSLEs), resulting in cells that are easily killed. This work investigated the regulation of the GSLE SleB by other proteins in the spore. A full understanding of how GSLEs are held inactive in the dormant spore and are activated during germination could lead to development of simplified spore decontamination strategies in which spore germination is the first step.
It was found that SleB and YpeB are co-dependent. In the absence of one protein, the other is degraded during sporulation by an unidentified protease(s), although HtrC and SpoIVB are not likely responsible. Specific regions and residues of YpeB were also identified as being important to its relationship with SleB. While some evidence suggests that SleB and YpeB physically interact, a direct interaction was not observed in vivo or in vitro. YpeB was demonstrated to be proteolytically processed by HtrC during germination, resulting in stable products containing the YpeB C-terminus. The presence of inhibitory PepSY domains at the C-terminus of YpeB, coupled with YpeB degradation during germination, may suggest that YpeB processing results in SleB activation. Modification of the predominant YpeB cleavage sites or
deletion of htrC reduced proteolysis, but cleavage at other sites still resulted in YpeB instability. Additionally, these changes did not have a significant impact on SleB activity.
SleB regulation by other spore proteins was also examined. To test if SleB activation is Ger receptor-dependent, Bacillus subtilis strains lacking Ger receptors and/or GSLEs were germinated via non-nutrient means. Results indicated SleB can be activated independent of these proteins. B. anthracis homologs of the B. subtilis lipoproteins YlaJ and YhcN were also studied, but deletion of these genes did not result in significant changes in SleB stability or activity.